FR3045408A1 - ASSEMBLIES OF HYDROPHOBIC NANOPARTICLES IN AQUEOUS MEDIA - Google Patents

Abstract

The present invention is in the field of nanomaterials. More particularly, the present invention relates to novel nano-objects, hybridosomes, consisting of an aqueous core and an organic-inorganic hybrid shell. This includes both inorganic nanoparticles and polymer chains. The present invention also relates to a process for preparing these hybridosomes and their uses.

Description

ASSEMBLIES OF HYDROPHOBIC NANOPARTICLES IN A MEDIUM

AQUEOUS

FIELD OF THE INVENTION

The present invention is in the field of nanomaterials. More particularly, the present invention relates to novel nano-objects, termed hybridosomes, consisting of an aqueous core and an organic-inorganic hybrid shell. This includes both inorganic nanoparticles and polymer chains. The present invention also relates to a process for preparing these hybridosomes, and the use of these nano-objects in various fields such as that of imaging, optics, plasmonics or in the biomedical field.

STATE OF THE ART

Nano-objects find their applications in a wide variety of fields, including paintings, optics, optoelectronics and biomedical applications such as medical imaging.

Among the diversity of existing materials, hybrid nano-objects of the type "inorganic nanoparticles / organic polymer" are able to combine their individual properties and to provide new materials with unique properties. These materials are therefore the subject of increased research.

So far, assemblies of hybrid nano-objects have been obtained according to three main strategies.

The first, in situ synthesis, uses polymeric, linear or hyperbranched architectures (Keilitz et al., Chem., Mater., 2008, 20, pp 2005-2007, Corr et al, J. Am., Chem., Soc. , 130, pp. 4214-4215). These promote the nucleation of the particle while avoiding the formation of uncontrolled aggregates. However, in situ synthesis generally leads to a relatively poor control of the morphology of the nanoparticles and / or their assemblies. Therefore, it is preferable, when high quality nanoparticles are expected, to dissociate synthesis from their assembly.

The second strategy relates to the encapsulation of preformed nanoparticles within cores of surfactants or micelles of amphiphilic copolymers (Rahme et al, ChemPhysChem, 2008, 9, pp 2230-2236, Hicket, RJ et al, J. Am Chem Soc., 133, pp 1517-1525). This technique provides more efficient control of assembly morphology but generally results in the formation of small aggregates (i.e., a small amount of nanoparticles in these assemblies).

The last strategy is to achieve simultaneous co-assembly of polymers and nanoparticles. In the case of nano-precipitation, an amount of a "bad" solvent is added to a polymer solution which leads to the formation of aggregates. For example, Casterou et al. (Chem Eur.J. 2015, 21, 1-8) have produced nano-objects comprising a core of magnetic particles and a bark of hydrophilic polymers from a water-tetrahydrofuran (THF) mixture by "solvophobic effect". . Its principle consists, in the first place, in solubilizing the magnetic nanoparticles in "good" solvent (i.e. in a solvent in which the nanoparticles are dispersed) such as THF. Then a small amount of a "bad" solvent (such as water) is added in order to destabilize the nanoparticles and to cause their aggregation. The last step is to stabilize the nanoparticle aggregates by adding polymer chains. However, nano-precipitation of nanoparticles, in particular by solvophobic effect, does not make it possible to obtain nano-objects having a homogeneous size distribution, which limits their applications. In addition, this strategy is not particularly aimed at obtaining a capsule - shell structure. Thus, it is not conceivable by this technique to obtain nano-objects having a heart; a fortiori a liquid heart.

Another technique, the emulsion, which involves a dispersion of an oil in water (or vice versa) in which the oil and water are immiscible, makes it possible to obtain nano-objects with a liquid heart. However, the objects obtained are generally larger than one micron and it is necessary to stabilize the formation of these objects throughout the process by the use of surfactants.

Therefore, the techniques known to date do not make it possible to manufacture submicron objects based on nanoparticles having a core-shell structure, by a process that is easy to implement. In particular, there is no known to the Applicant a method for providing stable nano-objects without surfactant, having an aqueous core. However, these objects could have many advantages, particularly in the biomedical field (absence of residual toxic organic solvent, possibility of accessing a wider choice of nanoparticles, etc.).

In this context, there is still a need to provide new synthetic routes for producing versatile hybrid nano-objects with good structural homogeneity.

Surprisingly, the Applicant has managed to develop a new process for the preparation of hybrid nano-objects. In particular, the Applicant has shown that it is possible, from a mixture of water and a solvent miscible in water, to form at an early stage stable drops of this solvent in water; and this, in the absence of surfactants.

The Applicant has also shown that these drops of organic solvent dispersed in the aqueous phase, can be used as a "template" to self-associate on their surface, inorganic nanoparticles and polymer chains. A simple removal of the solvent makes it possible to obtain nano-objects dispersed in an aqueous medium, these nano-objects consisting of a shell and an aqueous core.

The method of the invention has the advantage of being easy to implement. In addition, the use of drops of organic solvent as "template", allows to obtain unique nano-objects by their structure and having a uniform size distribution.

Against all expectations, the method of the invention also makes it possible to self-associate hydrophobic compounds in a predominantly aqueous medium, and this without these objects precipitating macroscopically. Thus, the nano-objects of the invention have very wide use capabilities ranging from the encapsulation of compounds, in particular hydrophobic compounds, to the use of the specific properties of these objects. SUMMARY The invention thus relates to a process for preparing nano-objects consisting of a shell and an aqueous core, called hybridosomes, comprising at least: (i) a step of forming stable spherical drops of an organic solvent in water in the absence of surfactants; said organic solvent and water being miscible; (ii) a step of self-assembly of hydrophobic inorganic nanoparticles on the surface of the spherical drops of the solution defined in (i); (iii) a step of adding polymer chains to the surface of the spherical drops obtained in (ii); (iv) a step of removing the organic solvent.

According to one embodiment, steps (i) and (ii) are performed simultaneously or concomitantly. According to one embodiment, steps (i) and (ii) are carried out successively.

According to one embodiment, the aqueous solution comprises from more than 0% to 30% vol. an organic solvent; preferably, the aqueous solution comprises 20% vol. an organic solvent.

According to one embodiment, the organic solvent is tetrahydrofuran (THF).

According to one embodiment, the removal of the organic solvent is carried out by evaporation. The invention also relates to a new nano-object, called a hybridosome, having: (a) an aqueous core having a volume of 500,000 to 40,000,000 nm3, (b) and a hybrid shell comprising: hydrophobic inorganic nanoparticles, functionalized or not; preferably, magnetic nanoparticles; and organic polymer chains; said hybridosome having a mean diameter in a range of 50 nm to 400 nm.

According to one embodiment, the inorganic nanoparticles are metallic nanoparticles. According to one embodiment, the inorganic nanoparticles are magnetic nanoparticles.

According to one embodiment, the hydrophobic inorganic nanoparticles are chosen from nanoparticles of iron oxides, gold nanoparticles, "quantum dots" or combinations thereof.

According to one embodiment, the organic polymer chains are chosen from hydrophilic and biocompatible polymer chains.

According to a crosslinking mode, the organic polymer chains are not crosslinked.

According to one embodiment, the organic polymer chains are chosen from poly (acrylic acid), poly (vinylpyrrolidone) or poly (ethyleneglycol) -co-poly (acrylic acid).

According to one embodiment, the hybridosomes of the present invention are useful in the field of imaging and / or in the biomedical field.

According to one embodiment, the hybridosomes of the present invention are useful as contrast agents.

DEFINITIONS

In the present invention, the terms below are defined as follows: "Alkyl": relates to an optionally substituted linear or branched hydrocarbon chain containing from 1 to 20 carbon atoms; preferentially the term alkyl includes methyl, ethyl, propyl, isopropyl, n-butyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl. "Hybrid hull": refers to a stable shell-type spherical assembly comprising at least one assemblage of inorganic compounds and at least one organic compound. For the purposes of the present invention, the inorganic compound relates to an assembly of inorganic nanoparticles; preferably, magnetic nanoparticles; preferably, metal nanoparticles, and the organic compound refers to one or more polymer chains having physical or chemical interaction with the inorganic nanoparticle assembly. "About": placed in front of a number, means plus or minus 10% of the nominal value of that number. "Hybridosome" relates to a nano-object comprising at least one hybrid shell and an aqueous core. "Nano-objects" relates to assemblies of nanoparticles. For the purposes of the present invention, nano-objects are stable spherical assemblies of inorganic nanoparticles. These nano-objects have a mean diameter in a range from 50 nm to 400 nm; preferably from 80 nm to 250 nm; more preferably, approximately equal to 105 nm. "Nanoparticle": relates to a particle having dimensions less than 50 nm; preferably, less than 20 nm. In the case of a spherical nanoparticle, the average diameter is less than 50 nm; preferably less than 20 nm; more preferably, approximately equal to 5 nm. DETAILED DESCRIPTION Process

The present invention relates to a process for preparing new nano-objects consisting of a shell and an aqueous core, called hybridosomes, comprising at least: (i) a step of forming stable spherical drops of an organic solvent in water in the absence of surfactants; said organic solvent and water being miscible; (ii) a step of self-assembly of hydrophobic inorganic nanoparticles on the surface of the spherical drops of the solution defined in (i); (iii) a step of adding polymer chains to the surface of the spherical drops obtained in (ii); (iv) a step of removing the organic solvent.

According to one embodiment, the method of the invention is not an emulsion process. According to one embodiment, the method of the invention is not a nano precipitation method.

According to one embodiment, steps (i) and (ii) are performed simultaneously or concomitantly. According to one embodiment, steps (i) and (ii) are carried out successively.

Etaye (i)

In one embodiment, the organic solvent is selected from tetrahydrofuran (THF); alcohols such as methanol, ethanol, propanol, butanol, tertbutanol, pentanol, isopropanol; acetone; dimethylsulfoxide (DMSO), dimethylformamide (DMF), acetonitrile; dioxane such as 1,2-dioxane, 1,3-dioxane or 1,4-dioxane or morpholine. According to one embodiment, the organic solvent miscible with water is THF.

According to one embodiment, the aqueous solution comprises from more than 0% to 30% vol. an organic solvent miscible with water relative to the total volume of the mixture; preferably from 5% to 25% vol. an organic solvent miscible with water; more preferably, the aqueous solution comprises about 20% vol. an organic solvent miscible with water.

According to one embodiment, the spherical drops formed in step (i) do not result from an emulsion. According to one embodiment, the spherical drops formed in step (i) are stable in the mixture. For the purposes of the present invention, the term "stable spherical drops" drops organic solvent that does not coalesce, even in the absence of surfactants or other additives.

According to one embodiment, the spherical drops formed in step (i) comprise a compound of interest chosen from a fluorophore or a pharmaceutically active agent; preferably, a compound of hydrophobic interest. According to one embodiment, the spherical drops formed in step (i) comprise bodipy (or boron-dipyromethene) or one of its derivatives. According to one embodiment, the spherical drops formed in step (i) comprise doxorubicin.

According to one embodiment, the spherical drops formed in step (i) have a mean hydrodynamic diameter, measured by dynamic light scattering (DDL), in a range from 50 nm to 400 nm; preferably from 80 nm to 250 nm. According to one embodiment, the spherical drops formed in step (i) have a mean hydrodynamic diameter (Dh) of approximately 105 nm. According to one embodiment, the spherical drops formed in step (i) have a mean hydrodynamic diameter (Dh) of about 140 nm.

According to one embodiment, step (i) is conducted at a temperature in a range from more than 0 ° C to 40 ° C; preferably from 15 ° C to 30 ° C; more preferably, step (i) is conducted at a temperature of about 25 ° C. According to one embodiment, step (i) is conducted at room temperature.

According to one embodiment, step (i) is conducted at ambient atmospheric pressure. According to one embodiment, step (i) is carried out at an atmospheric pressure of approximately 1 bar.

Step (ii)

According to one embodiment, step (ii) is conducted at a temperature in a range of greater than 0 ° C to 40 ° C; preferably from 15 ° C to 30 ° C; more preferably, step (ii) is conducted at a temperature of about 25 ° C. According to one embodiment, step (ii) is conducted at room temperature.

According to one embodiment, step (ii) is conducted at ambient atmospheric pressure. According to one embodiment, step (ii) is conducted at an atmospheric pressure of approximately 1 bar.

Inorganic nanoparticles

In one embodiment, the inorganic nanoparticles of step (ii) are chosen from metal nanoparticles, fluorescent nanoparticles such as "quantum dots", rare earth clusters, metal oxides, transition metal oxides. carbon particles such as "carbon dots" (Li et al., J. Mater Chem., 2012, 22, 24230), graphene oxides, fullerenes, graphene or their derivatives, and combinations thereof; preferably, gold, manganese, gadolinium, "quantum dots", iron oxides, tungsten oxides, titanium oxides, zinc oxides, cerium oxides and combinations thereof.

In the present invention, the term "quantum dots" colloidal nanoparticles semiconductors having a crystalline structure and fluorescence properties (Bera et al., Material, 2010, 3, pp 2260-2345).

According to one embodiment, the inorganic nanoparticles of step (ii) are iron oxide particles selected from y-FeiO 3. Fe3O4, CoFe2O4, MnFe2O4, CuFe2O4, NiFe2O4. According to one embodiment, the inorganic nanoparticles of step (ii) are maghemite particles y-Fc10. According to one embodiment, the inorganic nanoparticles of step (ii) are Fe304 magnetite particles. According to one embodiment, the inorganic nanoparticles are superparamagnetic nanoparticles. According to one embodiment, the inorganic nanoparticles are paramagnetic nanoparticles. According to one embodiment, the inorganic nanoparticles are gold nanoparticles. According to one embodiment, the inorganic nanoparticles are "quantum dots".

According to one embodiment, the inorganic nanoparticles of step (ii) are synthesized by techniques known to those skilled in the art including 1) physical methods such as aerosol or plasma methods; 2) chemical methods from inorganic precursors including co-precipitation, sol-gel method, microemulsion, solvothermal methods or organometallic methods; or else 3) biological methods.

According to one embodiment, the inorganic nanoparticles of step (ii) are of commercial origin.

According to one embodiment, the inorganic nanoparticles of step (ii) are obtained by solvothermal methods.

According to one embodiment, the inorganic nanoparticles of step (ii) are functionalized by alkyl chains that can be optionally functionalized with thiol functions, amines, phosphates, phosphonic acids, carboxylic acids; preferably, the inorganic nanoparticles of step (ii) are covered with dodecanethiol or octylamine. According to one embodiment, the inorganic nanoparticles of step (ii) are not functionalized with silica. According to one embodiment, the inorganic nanoparticles of step (ii) are not covered with silica. According to one embodiment, the inorganic nanoparticles of step (ii) are covered with silica. According to one embodiment, the inorganic nanoparticles of step (ii) are covered with silica functionalized with hydrophobic ligands.

According to one embodiment, the inorganic nanoparticles of step (ii) are functionalized by polymer chains comprising pendant alkyl chains.

According to one embodiment, the inorganic nanoparticles of step (ii) have an average diameter of less than 50 nm; preferably, the inorganic nanoparticles of step (ii) have an average diameter of less than 20 nm; more preferably, the inorganic nanoparticles of step (ii) have an average diameter of approximately 5 nm.

According to one embodiment, the inorganic nanoparticles of step (ii) have a spherical shape. According to one embodiment, the nanoparticles are not in the form of nanotubes or rods. According to one embodiment, the inorganic nanoparticles of step (ii) are not aggregated. According to one embodiment, the inorganic nanoparticles of step (ii) have a cubic, parallelepipedic, star shape or are in the form of nano-tubes or rods.

According to one embodiment, during self-assembly of the inorganic nanoparticles of step (ii), the inorganic nanoparticles partially or totally cover the surface of the spherical drops obtained in step (i). According to one embodiment, during the self-assembly of the inorganic nanoparticles of step (ii), the inorganic nanoparticles partially cover the surface of the spherical drops obtained in step (i). According to one embodiment, during the self-assembly of the inorganic nanoparticles of step (ii), the inorganic nanoparticles totally cover the surface of the spherical drops obtained in step (i). According to one embodiment, during the self-assembly of the inorganic nanoparticles of step (ii), the inorganic nanoparticles homogeneously cover the surface of the spherical drops obtained in step (i).

Advantageously, the method of the present invention makes it possible to self-assemble inorganic nanoparticles by strong adsorption on the surface of the spherical drops obtained in step (i).

According to one embodiment, the molar concentration of metal atoms introduced into the medium during step (ii) is less than 1.5 mmol / L; preferably from 0.35 to 1 mmol / L; more preferably, the amount of metal atoms introduced into the medium during step (ii) is approximately equal to 0.47 mmol / L. According to one embodiment, the molar concentration of iron atoms introduced into the medium during step (ii) is less than 1.5 mmol / L; preferably from 0.35 to 1 mmol / L; more preferably, the molar concentration of iron atoms introduced into the medium during step (ii) is approximately equal to 0.47 mmol / L.

Etaye (iii)

According to one embodiment, step (iii) is conducted at a temperature in a range of greater than 0 ° C to 40 ° C; preferably from 15 ° C to 30 ° C; more preferably, step (iii) is carried out at a temperature of about 25 ° C. According to one embodiment, step (iii) is conducted at room temperature.

According to one embodiment, step (iii) is conducted at ambient atmospheric pressure. According to one embodiment, step (iii) is carried out at an atmospheric pressure of approximately 1 bar.

Polymer chains

In one embodiment, the polymer chains are selected from homopolymers or copolymers of hydrophilic polymers. According to one embodiment, the polymer chains are chosen from homopolymers or copolymers of biocompatible polymers. According to one embodiment, the polymer chains are poly (acrylic acid) chains, PAA or their derivatives. According to one embodiment, the polymer chains are polyphosphonate chains or their derivatives. According to one embodiment, the polymer chains are polyamine chains or their derivatives. According to one embodiment, the polymer chains are chains of poly (vinylpyrrolidone), PVP or their derivatives. According to one embodiment, the polymer chains are polyolefin chains. According to one embodiment, the polymer chains are chains of poly (ethylene glycol), PEG or their derivatives. According to one embodiment, the polymer chains are copolymers of poly (ethylene glycol) and poly (acrylic acid); also noted poly (ethylene glycol) -co-poly (acrylic acid), (PEG-PAA). According to one embodiment, the polymer chains are poly (amino acids) or polypeptides, or their derivatives; preferably homopolymers or copolymers of poly (lysine) or poly (cysteine). According to one embodiment, the polymer chains are poly (siloxanes) or their derivatives. According to one embodiment, the polymer chains are poly (styrene) or their derivatives; preferably, polystyrene sulfonic acids. According to one embodiment, the polymer chains are polyvinyl alcohol. According to one embodiment, the polymer chains are biological macromolecules; preferably, polysaccharides or proteins. In one embodiment, the polymer chains are deoxyribonucleic acid (DNA) strands. According to one embodiment, the polymer chains are collagen chains. According to one embodiment, the polymer chains are conductive polymer chains; preferably polythiophene chains or chains of poly (3,4-ethylenedioxythiophene) -co-polystyrene sulfonate (PEDOT-PSS), and derivatives thereof.

According to one embodiment, the polymer chains have monobloc, diblock or triblock structures.

According to one embodiment, the polymer chains are linear chains or branched chains. The term "branched chains" means any linear polymer chain having at least one branch point on which is fixed a pendant side chain. Unlike crosslinked chains, branched polymer chains are not three-dimensional networks. Advantageously, the method of the present invention does not require crosslinking the polymer chains to stabilize the self-assemblies of inorganic nanoparticles.

According to one embodiment, the polymer chains are synthesized according to the techniques known to those skilled in the art, including, in particular, radical polymerization, polycondensation or polyaddition polymerization techniques. According to one embodiment, the polymer chains are of commercial origin.

According to one embodiment, the polymer chains have a molecular weight ranging from 500 Da to 2000 kDa; preferably from 1 kDa to 1500 kDa. According to one embodiment, the polymer chains have a molecular weight of approximately 1.8 kDa, 450 kDa or 1250 kDa.

According to one embodiment, the polymer chains are adsorbed on the inorganic nanoparticles. According to one embodiment, the polymer chains form with the inorganic nanoparticles, a hybrid shell. According to one embodiment, the polymer chains are distributed homogeneously on the surface of the spherical drops of solvent obtained in step (i).

According to one embodiment, the polymer chains may be functionalized with peptides; proteins; antibodies; kinase inhibitors; fluorophores; complexes of radioelements. According to the method of the invention, the hybrid shell of the nano-objects can easily be functionalized by changing the nature or the functionalities of the polymer chains.

Step (iv)

According to one embodiment, the removal of the organic solvent is done by evaporation. For the purposes of the present invention, the elimination of the solvent after the addition of polymer chains during step (iii) makes it possible to obtain a suspension of hydrophobic nanoobjects in an aqueous medium. More particularly, the step of removing the organic solvent after the formation of the hybridosomes by the method of the invention as described above, allows the encapsulation of hydrophobic compounds in aqueous medium. According to one embodiment, the compounds of interest encapsulated in the hybridosome are not inorganic nanoparticles; in particular, magnetic nanoparticles. According to one embodiment, the compounds of interest encapsulated in the hybridosome are not metal nanoparticles.

According to one embodiment, the removal of the organic solvent is carried out by evaporation. According to one embodiment, the step of evaporation of the organic solvent is conducted at a temperature of more than 30 ° C; preferably, the step of evaporation of the organic solvent is carried out at a temperature of approximately 43 ° C.

According to one embodiment, the removal of the solvent is done by dialysis.

According to one embodiment, the step of removing the organic solvent is carried out at ambient atmospheric pressure. According to one embodiment, the step of removing the organic solvent is carried out at an atmospheric pressure of approximately 1 bar.

According to one embodiment, the step of evaporation of the organic solvent is carried out slowly; preferably, the evaporation step of the organic solvent is conducted for about 15h.

According to one embodiment, the step of removing the organic solvent is carried out slowly; preferably, the organic solvent removal step is conducted for about 15h.

According to one embodiment, the method of the invention provides a spherical nano-object comprising a hybrid shell and a hollow core, called a hybridosome, which can optionally comprise compounds of interest, in particular hydrophobes. According to one embodiment, the compounds of interest encapsulated in the hybridosome do not fill the entire hollow core.

In one embodiment, the method of the invention further comprises washing steps of the nano-objects. According to the method of the invention, the washing steps can be carried out by any technique known to those skilled in the art such as centrifugation, magnetic separation or dialysis. According to a preferred embodiment, the washing steps are carried out by magnetic separation.

Hybridosomes The invention also relates to a novel nano-object, called a hybridosome, having: (a) an aqueous core having a volume of 500,000 to 40,000,000 nm3; (b) and a hybrid shell comprising: hydrophobic inorganic nanoparticles, functionalized or otherwise; preferably, magnetic nanoparticles; and organic polymer chains; said hybridosome having a mean diameter in a range of 50 nm to 400 nm; preferably from 80 nm to 250 nm; more preferably, approximately equal to 105 nm. Aqueous heart

In one embodiment, the heart of the hybridosome does not consist of a solid monophase. In particular, within the meaning of the present invention, the heart of the hybridosome is neither a gel nor a solid. According to one embodiment, the heart of the hybridosome does not include internal pores. According to one embodiment, the heart of the hybridosome does not comprise silica or its derivatives.

According to one embodiment, the heart of the hybridosome is liquid: more particularly aqueous.

According to one embodiment, the core consists of a liquid phase comprising compounds of interest chosen from fluorophores or pharmaceutically active agents. For the purpose of the present invention, the encapsulation of compounds of interest does not lead to the filling of the entire volume defined by the hybrid shell of the nano-objects. According to one embodiment, the interest compounds encapsulated in the core of the nanoobjects of the invention may be in the form of a suspension of individualized elements or nano-aggregates.

According to one embodiment, the heart of the hybridosome does not include surfactants. According to one embodiment, the heart of the hybridosome does not comprise cytotoxic solvents.

Hybrid hull

In one embodiment, the hybrid shell consists of inorganic nanoparticles and organic polymer chains.

According to one embodiment, the inorganic nanoparticles are chosen from metal nanoparticles, fluorescent nanoparticles such as "quantum dots", rare earth clusters, metal oxides, transition metal oxides, carbon particles such as carbon dots, graphene oxides, fullerenes, graphene or their derivatives, and combinations thereof; preferably, gold, manganese, gadolinium, "quantum dots", iron oxides, tungsten oxides, titanium oxides, zinc oxides, cerium oxides and combinations thereof.

According to one embodiment, the inorganic nanoparticles are iron oxide particles chosen from y-FeiO 3. Fe3O4, CoFe2O4, MnFe2O4, CuFe2O4, NiFe2O4. According to one embodiment, the inorganic nanoparticles are particles of maghemite γ-Fe2C> 3. According to one embodiment, the inorganic nanoparticles are Fe304 magnetite particles. According to one embodiment, the inorganic nanoparticles are superparamagnetic nanoparticles. According to one embodiment, the inorganic nanoparticles are paramagnetic nanoparticles. According to one embodiment, the inorganic nanoparticles are gold nanoparticles. According to one embodiment, the inorganic nanoparticles are "quantum dots". According to one embodiment, the inorganic nanoparticles are nanoparticles of manganese. In one embodiment, the inorganic nanoparticles are gadolinium nanoparticles.

According to one embodiment, the inorganic nanoparticles are functionalized by alkyl chains which can be optionally functionalized by thiol functions, amines, phosphates, phosphonic acids, carboxylic acids; preferably, the inorganic nanoparticles are coated with dodecanethiol. According to one embodiment, the inorganic nanoparticles are functionalized with octylamine or dodecylamine. According to one embodiment, the inorganic nanoparticles are not functionalized with silica. According to one embodiment, the inorganic nanoparticles are not covered with silica. According to one embodiment, the inorganic nanoparticles are covered with silica.

According to one embodiment, the inorganic nanoparticles are functionalized by polymer chains comprising pendant alkyl chains. According to one embodiment, the inorganic nanoparticles are functionalized by poly (amino acid) chains. According to one embodiment, the inorganic nanoparticles are functionalized with peptides.

According to one embodiment, the inorganic nanoparticles have an average diameter of less than 50 nm; preferably, the inorganic nanoparticles have an average diameter of less than 20 nm; more preferably, the inorganic nanoparticles have an average diameter of approximately 5 nm.

According to one embodiment, the inorganic nanoparticles have a spherical shape. According to one embodiment, the nanoparticles are not in the form of nanotubes or rods. According to one embodiment, the inorganic nanoparticles are not aggregated. According to one embodiment, the inorganic nanoparticles have a cubic, parallelepiped or star shape.

According to one embodiment, the inorganic nanoparticles self-associate in the form of a spherical shell having a mean diameter in a range from 50 nm to 400 nm; preferably from 80 nm to 250 nm; more preferably, approximately equal to 105 nm.

In one embodiment, the polymer chains are selected from homopolymers or copolymers of hydrophilic polymers. According to one embodiment, the polymer chains are chosen from homopolymers or copolymers of biocompatible polymers. According to one embodiment, the polymer chains are poly (acrylic acid) chains, PAA or their derivatives. According to one embodiment, the polymer chains are polyphosphonate chains or their derivatives. According to one embodiment, the polymer chains are polyamine chains or their derivatives. According to one embodiment, the polymer chains are chains of poly (vinylpyrrolidone), PVP or their derivatives. According to one embodiment, the polymer chains are polyolefin chains. According to one embodiment, the polymer chains are chains of poly (ethylene glycol), PEG or their derivatives. According to one embodiment, the polymer chains are copolymers of poly (ethylene glycol) and poly (acrylic acid); also noted poly (ethylene glycol) -co-poly (acrylic acid), (PEG-PAA). According to one embodiment, the polymer chains are poly (amino acids) or polypeptides, or their derivatives; preferably homopolymers or copolymers of poly (lysine) or poly (cysteine). According to one embodiment, the polymer chains are poly (siloxanes) or their derivatives. According to one embodiment, the polymer chains are poly (styrene) or their derivatives; preferably, polystyrene sulfonic acids. According to one embodiment, the polymer chains are polyvinyl alcohol. According to one embodiment, the polymer chains have monobloc, diblock or triblock structures.

According to one embodiment, the polymer chains are linear chains or branched chains.

According to one embodiment, the polymer chains are not crosslinked chains.

According to one embodiment, the polymer chains have a molecular weight ranging from 500 Da to 2000 kDa; preferably from 1 kDa to 1500 kDa. According to one embodiment, the polymer chains have a molecular weight of approximately 1.8 kDa, 450 kDa or 1250 kDa.

According to one embodiment, the polymer chains are adsorbed on the inorganic nanoparticles. According to one embodiment, the polymer chains form with the inorganic nanoparticles, a hybrid shell

Hybridosomes obtained by the process The invention also relates to a novel nano-object, called a hybridosome, obtainable by the method of the invention as described above.

uses

The present invention also relates to the use of the nano-objects of the invention in the field of optics, plasmonics, imaging or the biomedical field including theragnostics.

According to one embodiment, the hybridosomes as defined above can be used for biological separation.

According to one embodiment, the hybridosomes as defined above can be used in hyperthermia therapy.

According to one embodiment, the hybridosomes as defined above, can be used as contrast agents; preferably, magnetic resonance imaging (MRI).

According to one embodiment, the hybridosomes as defined above, can be used for any type of imaging such as fluorescence imaging, imaging involving X-rays, ultrasound, magnetic field or radioactivity natural or artificial.

BRIEF DESCRIPTION OF THE FIGURES

Figure IA is a stability diagram for different water / THF mixtures as a function of the water volume fraction (Φ) in the medium.

Figure IB is a photograph of self-assemblies of iron nanoparticles observed by SEM.

Figure 2 is a graph showing the size distribution measured by "Nanoparticle Tracker", drops of THF formed in a THF / water mixture for a water volume fraction Φ equal to 0.8.

Figure 3A is a graph showing the size distribution measured by "Nanoparticle Tracker", IONP @ PAA hybridosomes according to the concentration of particles in the medium.

Figure 3B is a photograph of IONP @ PAA hybridosomes observed by MET.

Figure 4 is a photograph of a self-assembly of nanoparticles of gold and iron observed by MET.

Figure 5 is a graph showing the absorbance and emission spectra of IONP / Bodipy @ PAA hybridosomes as a function of wavelength.

Figure 6 is a T2-weighted image of a mouse liver a) before injection, b) after injection at t = to + 30 min and c) after injection at t = to + 24h, hybridosomes IONP @ PEG-PAA.

EXAMPLES

The present invention will be better understood on reading the following examples which illustrate the invention in a nonlimiting manner.

Abbreviations

Bodipy: boron-dipyromethene ° C: degree Celsius

Dh: hydrodynamic diameter DDL: Dynamic scattering of light (Diffusion Light Scattering, DLS) IONP: Iron oxide particles (Iron Oxide nanoparticle, IONP)

IONP @ PAA: Hybridosomes comprising a hybrid shell consisting of an autoassembly of iron nanoparticles and PAA chains mL: milli-liter PAA: poly (acrylic acid) PEG-PAA: copolymer of poly (ethylene glycol) and poly (acrylic acid) PVP: poly (vinylpyrrolidone) MET: transmission electron microscopy THF: tetrahydrofuran

Materials and methods Reagents

All chemical reagents and solvents were obtained from commercial sources (Sigma Aldrich or VWR) and were used as such. The water used as a solvent is ultra-pure water obtained via the Elga PURELAB system. Gold particles coated with dodecanthiol (AuNPs) were purchased from nanoComposix and redispersed in THF. The polymers used in the examples relate to poly (acrylic acid) (1.8 kDa, 450 kDa or 1250 kDa) and poly (vinylpyrrolidone) (360 kDa).

Dynamic light scattering, DDL (Dynamic Light Scattering, DLS)

The particle sizes and their distribution were evaluated by measuring the hydrodynamic diameter (Dh) with a Zeta-sizer nano-ZS device from Malvem Instruments SA combined with a laser operating at 633 nm.

Transmission electron microscopy (TEM)

The samples were deposited on 200mesh copper grids coated with a carbon / formvar film. The observations were made using a JEOL 1400 operating at 120kV and equipped with a GATAN Orius 1000 camera.

Scanning electron microscopy (SEM)

A few microliters of sample were deposited on a piece of polished silicon substrate. The observation was carried out using a JEOL 7100f microscope equipped with a tip-type tungsten source and an Everhart-Thomley type detector operating at voltages between 5 and 10kV.

Nanoparticle Tracking Analysis (Nanoparticle Tracker Analysis NTA)

The size, distribution, and concentration of the objects were also evaluated by analyzing the individual tracking of the Brownian particle motion. The samples were introduced into the measuring chamber of a Nanosight LM10 from Malvem Instruments SA equipped with a 405 nm wavelength exciting laser and a Scientific CMOS Image Sensor camera.

Magnetic Resonance Imaging (MRI)

Hybridosomes were characterized by MRI using a 4.7 T preclinical Brucker device. The hybridosomes were injected intravenously and the T2 * weighted images were acquired before injection, 30 minutes and 24h after injection with the patients. sequence parameters: TE = 5ms, TR = 300ms, flip angle = 30 °.

UV-Vis spectroscopy

UV-Visible absorbance spectra were measured on a Cary 60 UV-Vis apparatus from Agilent Technologies.

Fluorescence spectroscopy

The fluorescence emission spectra were measured on an apparatus of Edinburgh Instruments (FLS920) equipped with a 450W Xenon lamp and a photon counter type detector. A. Synthesis of magnetic nanoparticles

Example 1 Synthesis and Characterization of Magnetic Nanoparticles of Iron Oxide QONP)

The synthesis of IONP particles was conducted via the hydrolysis of an organometallic precursor of iron (II) [Fe (N (SiMe3) 2) 2; 188 mg; 0.5 mmol] in the presence of 2 equivalents of a stabilizing ligand, octylamine, according to the procedure described in Glaria et al., Chem. Phys. Chem., 2008, 9, pp776-780 and pp2035-2041.

The IONP particles were characterized by MET and DDL. The average diameter of the nanoparticles is approximately equal to 5.6 nm.

B. Assembly of Hydrophobic Nanoparticles in Various Water / THF Mixtures

Example 2 Influence of the volume fraction in water

Three water / THF / nanoparticles mixtures of hydrophobic iron oxides (IONPs) were prepared with volume fractions in water respectively equal to 0; 0.42 and 0.80. The hydrophobic iron oxide nanoparticles (IONPs) have an average diameter of approximately 5.5 nm.

The purpose of this experiment is to determine for each mixture: 1) whether the NPs are stable in suspension in the water / THF mixture (that they do not form aggregates which sediment in the mixture); and 2) if the assemblies of NPs form spherical objects.

These mixtures were characterized by measuring the quantity of nanoparticles in the supernatant by a measurement of the absorbance in UV on the one hand, and by MET on the other hand.

The results presented in FIG. 1 show that: in the absence of water (Φ = 0), 100% of the IONPs particles introduced into the medium are contained in the supernatant. This means that no aggregation or sedimentation of the IONPs particles is observed when the medium consists of an organic solvent in the absence of water; when the medium contains moderate amounts of water (Φ = 0.05 to 0.4) which is a poor solvent for IONPs hydrophobic nanoparticles, they rapidly aggregate and lead, as expected, to sedimentation of the objects; on the other hand, for mixtures consisting mainly of water (Φ> 0.7) for which it is expected that the hydrophobic NPs sediment, the most stable assemblies are obtained. In addition, the TEM photographs show that these nanoparticles self-assemble in a perfectly spherical form. The average diameter of these assemblies is about 105 nm.

FIG. 1B presents a SEM image of a self-assembly of iron nanoparticles obtained in a THF-water mixture having a water volume fraction equal to 0.8.

In conclusion, these experiments highlight a new method of spherical self-assembly of nanoparticles from a water-THF mixture without the use of an oily phase and in the absence of surfactant. Contrary to the prejudices of those skilled in the art, it is also demonstrated the possibility of forming assemblies of hydrophobic nanoparticles in predominantly aqueous medium. These assemblages are perfectly organized in the form of spheres and remain stable over time. C. Demonstration of the formation of stable drops of THF in a THF / water mixture

The results presented in Example 2 led the Applicant to question the conditions for obtaining spherical self-assemblies of nanoparticles from a water-THF mixture.

An experiment was therefore conducted to prove the preformation of drops of THF in a water-THF mixture having a water volume fraction greater than 0.7.

A water-THF mixture with a water volume fraction Φ equal to 0.8 was prepared and analyzed by the Nanoparticle Tracker.

This instrument consists in sending a laser beam on a liquid sample. The particles suspended in the sample, if they exist, are fdmées by one or more cameras and the hydrodynamic diameter of the nanoparticles is calculated by the software "Nanoparticle Tracking Analysis" (NTA) of the company MALVERN.

Surprisingly, the Applicant observed drops in the water-THF mixture having a volume fraction in water Φ equal to 0.8. Figure 2 shows the size distribution of these drops formed within the mixture. The hydrodynamic diameter of the drops of THF in the mixture is approximately equal to 146 nm.

The Applicant has therefore demonstrated the preformation of drops of THF in a water-THF mixture for volume fractions in water greater than 0.7; and this, in the absence of surfactant. D. Synthesis of hybridosomes

Example 3: Synthesis and characterization of IONP @ 50pL polymer hybridosomes of hydrophobic IONP nanoparticles were suspended in THF at an iron concentration of 131.5 μg / ml. Then 800 μl of water was added in order to obtain a water / THF mixture with a volume fraction of water equal to about 0.8. The sample was vortexed a few seconds. The molar concentration of iron in the medium is approximately equal to 0.47 mmol / L.

After 12 hours, 50 μl of an aqueous solution of polymer (PAA or PEG-PAA at a concentration of approximately 3.4 mg / ml) is added to the water / THF mixture containing the IONP, described above. The mixture is then placed at 43 ° C overnight to evaporate the THF.

Finally, magnetic separation is performed (NeFeB 0.35 T Magnet) to remove the excess polymer. The hybridosomes are resuspended in 930 μl of water.

IONP @ PAA hybridosomes were characterized by "Nanoparticle Tracker" (Figure 3A) and MET (Figure 3B).

Figure 3A shows the size distribution of the obtained hybridosomes. It is found that the average size of the hybridosomes, approximately equal to 135 nm, is very close to the size of the drops of THF previously formed in the water-THF mixture (see Example 2 and Figure 2, 146 nm). Therefore, the final size of the hybridosomes is dependent on the preformation of THF drops in the medium. These constitute a "template" for the self-assembly of inorganic nanoparticles and then polymer chains in the medium.

Figure 3B shows that the hybridosomes are spherical in shape and consist of an assembly of individual inorganic nanoparticles.

EXAMPLE 4 Synthesis and Characterization of AuNP @ polymer, IQNP / AuNP @ polymer or IQNP / QD @ polymer Hybridosomes

The synthesis of hybridosomes from gold nanoparticles (AuNP), a mixture of iron oxide particles and gold (IONP / AuNP) or a mixture of iron oxide particles and "Quantum dots" (IONP / QD) was conducted according to a protocol similar to that previously described in Example 3.

Figure 4 is a photograph of a self-assembly of nanoparticles of gold and iron observed by MET. Their average diameter is approximately equal to 77 nm.

Experiment 5: Synthesis and characterization of hvbridosomes encapsulating a fluorophore

The synthesis of hybridosomes comprising a fluorophore was conducted according to a protocol similar to that previously described in Example 2. The fluorophore used is bodipy.

A solution of bodipy was prepared in THF at a concentration of 1 mM. The bodipy (100 μL, 0.1 pmol) was added to 100 μL of a suspension of IONP particles in THF at a concentration of 500 μg / mL. The mixture was vortexed for a few seconds and then 800 μl of water were added in order to obtain a water / THF mixture with a water volume fraction of approximately 0.8. The final iron concentration is about 0.47 mM. An aqueous solution of polymer is then added to the rectional medium. The mixture is then placed at 43 ° C overnight to evaporate the THF. Finally, several magnetic separation wash steps were performed (NeFeB 0.35 T Magnet) to remove excess polymer and bodipy. IONP / Bodipy @ polymer hybridosomes were obtained. They were characterized by absorbance spectrometry and emission (Figure 5). The IONP / Bodipy @ polymer hybridosomes have an absorption maximum at a wavelength of approximately 535 nm.

Therefore, the formation of hybridosomes according to the method of the invention also makes it possible to encapsulate active compounds such as fluorophores. E. Uses of hybridosomes

Experiment 6: In Vivo MRI Imaging Test in Mice The efficacy of hybridosomes as MRI contrast agents was evaluated in vivo in the mouse model.

In this experiment, the study relates to hybridosomes consisting of an assembly of iron nanoparticles and PEG-PAA polymer chains (IONP @ PEG-PAA).

Prior to injection of the hybridosomes, an MRI image is taken from the liver of a mouse that has not received an injection (FIG. 6a). At 0 min, a quantity of hybridosomes is injected into the liver of the mouse. At t = + 30 min, a second MRI scan is taken from the mouse liver (Figure 6b). At t = to + 24h, a final MRI scan is taken from the liver of the mouse (Figure 6c).

The comparison of MRI images of the liver of the mouse before and after injection demonstrates the possible use of the hybridosomes of the invention as contrast agents.

Claims (10)

A process for preparing nano-objects consisting of a shell and an aqueous core, called hybridosomes, comprising at least: (i) a step of forming stable spherical drops of an organic solvent in water lack of surfactants; said organic solvent and water being miscible; (ii) a step of self-assembly of hydrophobic inorganic nanoparticles on the surface of the spherical drops of the solution defined in (i); (iii) a step of adding polymer chains to the surface of the spherical drops obtained in (ii); (iv) a step of removing the organic solvent. The process for preparing hybridosomes according to claim 1, wherein the aqueous solution comprises from more than 0% to 30% vol. an organic solvent; preferably, the aqueous solution comprises 20% vol. an organic solvent. A process for preparing hybridosomes according to claim 1 or claim 2, wherein the organic solvent is tetrahydrofuran (THF). 4. A method of preparing a hybridosome according to any one of claims 1 to 3, wherein the removal of the organic solvent is carried out by evaporation. 5. Nano-object, called a hybridosome, having: (a) an aqueous core having a volume of 500,000 to 40,000,000 nm3; (b) and a hybrid shell comprising: hydrophobic inorganic nanoparticles, functionalized or not; preferably, magnetic nanoparticles; and organic polymer chains; said hybridosome having a mean diameter in a range of 50 nm to 400 nm. Hybridosome according to claim 5, wherein the hydrophobic inorganic nanoparticles are selected from iron oxide nanoparticles, gold nanoparticles, quantum dots or combinations thereof. The hybridoma according to claim 5 or claim 6, wherein the organic polymer chains are selected from hydrophilic and biocompatible polymer chains. 8. Hybridosome according to claim 7, wherein the organic polymer chains are selected from poly (acrylic acid), poly (vinylpyrrolidone) or poly (ethyleneglycol) -co-poly (acrylic acid). 9. Use of the hybridosomes according to claims 5 to 8, in the field of imaging and / or in the biomedical field. 10. Use of the hybridosomes according to claim 9 as contrast agents.